RESUMEN
We formulated a selective enrichment broth (SVV) for simultaneous growth of Salmonella, Vibrio parahaemolyticus, and Vibrio cholerae by single factor experiment and response surface method. We evaluated the enrichment effect of SVV by conventional culture method and real-time PCR assay. We obtained the SVV broth by supplementting the Buffered Peptone Water (BPW) with bile salt no. 3, potassium tellurite, and sodium citrate as inhibitors, and glucose, mannitol, snhydrous sodium sulfite and sodium pyruvate as accelerants. We also modified the concentration of sodium chloride in BPW. When mixed at equal or varied proportions, the target pathogens had a great accumulation (10(5)-10(8) CFU/mL) after incubated in SVV for 18 h at 37 degrees C with shaking. It can also effectively inhibit the competitive microflora. We detected 10 artificial simulated samples and 608 real samples using SVV with real-time PCR. After enriched in SVV for 18 h, the quantity of the bacteria in samples were above the detection limit. The SVV with PCR assay showed higher tested positive (4.06%) compared to that of the conventional detection method (3.78%) and there was no false report. In summary, SVV is a promising new multiplex selective enrichment broth that can be used in detection of seafood.
Asunto(s)
Medios de Cultivo , Microbiología de Alimentos , Salmonella , Vibrio cholerae , Vibrio parahaemolyticusRESUMEN
Pseudomonas aernginosa (PA) is one of the most universal pathogens in clinical diagnosis, and conventional detection assay has many disadvantages. In this research, a pair of specific primers and a TaqMan fluorescent probe were designed in the conservative region of ETA gene by the method of bioinformatics analysis, the detection method for PA was successfully developed. Different gradient concentrations of PA DNA and various pathogen DNA were amplified by fluorescence quantitative PCR (FQ-PCR) to confirm the specificity and sensitivity of the developed method. Results showed that the developed detection assay is more sensible and specific by comparison to the conventional FQ-PCR method, and it is valuable for research and application prospects.
Asunto(s)
ADP Ribosa Transferasas , Genética , Toxinas Bacterianas , Genética , ADN Bacteriano , Exotoxinas , Genética , Colorantes Fluorescentes , Fluorometría , Métodos , Reacción en Cadena de la Polimerasa , Métodos , Pseudomonas aeruginosa , Genética , Sensibilidad y Especificidad , Polimerasa Taq , Factores de Virulencia , GenéticaRESUMEN
<p><b>UNLABELLED</b>We designed a pair of specific primers and a TaqMan fluorescent probe targeting the toxR gene of Vibrio parahaemolyticus (VP). After optimizing the conditions, the specialty, sensitivity and reproducibility of the detection method were evaluated.</p><p><b>RESULTS</b>(1) the developed real-time PCR assay protocol detected only VP and was not affected by other normal food pathogens such as Staphylococcus aureus, Salmonela, Listeria monocytogenes. (2) the limit of detection was 25 copies of toxR gene in the detected samples, and the sensitivity of pure cultures and simulated food samples was 21 cfu/mL and 210 cfu/g. (3) the developed protocol of real-time PCR assay had a high reproducibility, and the sample's variation was 0.9% and 1.3% within the same sample and between tests. (4) the standard curve had a good linearity when the gene quantity was between 2.5x10(1) and 2.5x10(6) copies. The developed detection assay targeting the toxR gene can quantitatively detect VP in only 3 hours, and thus is an efficacious method for the detection of Vibrio parahaemolyticus.</p>